淀粉酶产色链霉菌1628中toyF基因的克隆与丰加霉素合成相关性

Cloning of toyF gene and Its Relevance to Toyocamycin Biosynthesis in Streptomyces diastatochromogenes 1628

为考察编码腺苷琥珀酸裂解酶toyF基因对淀粉酶产色链霉菌Streptomyces diastatochromogenes 1628合成丰加霉素的影响,本研究设计简并引物,首次克隆获得了菌株1628的toyF基因,并构建了敲除质粒p KC1132-toyF’,通过接合转移转入菌株1628,获得toyF基因缺失株1628-ΔTOYF。结果表明,与原始菌株1628相比,缺失株1628-ΔTOYF中TOYF的酶活和丰加霉素产量分别降低66.7%、87.5%。在缺失株1628-ΔTOYF中回补表达toyF基因构建了重组菌1628-ΔTOYF/toyF。重组菌1628-ΔTOYF/toyF中的TOYF酶活和丰加霉素产量,较缺失株1628-ΔTOYF分别提高了2.7和8.3倍。由此可见,toyF基因是参与菌株1628生物合成丰加霉素的关键酶基因。本文toyF基因的克隆及其功能研究,为克隆完整的丰加霉素生物合成基因簇及其丰加霉素生物合成机制的研究奠定了基础。

To elucidate the ruction of toyF gene which encoded adenylosuccinate lyase in biosynthesis of toyocamycin （TM） in Streptomyces diastatochromogenes 1628, the toyF gene was successful cloned from S. diastatochromogenes 1628 by using degenerate primer. Then, knock-out plasmid pKC1132-toyF was constructed and integrated into the chromosome of S. diastatochromogenes 1628 by using intergeneric conjugation to yield strain 1628-ATOYF in which the toyF gene was disrupted. Both TOYF activity and TM production of in strain 1628-ATOYF decreased 66.7% and 87.5% comparing to those of wild-type strain S. diastatochromogenes 1628, respectively. Moreover, in order to express the toyF gene in strain 1628-ATOYF as complementary experiment, recombinant strain 1628-ATOYF/toyF was constructed. As a result, the recombinant strain 1628-ATOYF/toyF showed an increase of 2.7-fold in specific enzyme activity of TOYF and an increase of 8.3-fold in TM comparing to those of strain 1628-ATOYF, respectively. These results suggested that cloned toyF was a key gene which is involved in biosynthesis of TM in S. diastatochromogenes 1628. The cloning and functional analysis of toyF gene will pave the way for cloning the whole gene cluster which required for TM formation. Also, this work provides a basis for studying biosynthetic mechanism of TM in Streptomyces diastatochromogenes 1628.